1. Field of the Invention
The present invention relates to albumin-dye complexes and the quantitative determination of free fatty acids, compounds which upon hydrolysis yield fatty acids in aqueous systems, or in determination of the activity of hydrolase enzymes which will act on appropriate substrates to produce fatty acids.
2. Description of the Prior Art
It is known that albumin can bind a number of ligands, including dyestuffs, see for instance the disclosure by Oscar Bally in U.S. Pat. No. 1,543,543 and by Saturo Honio et al, in U.S. Pat. No. 3,679,586, Klotz et al in the Journal of the American Chemical Society, volume 68, 1946, pages 1486-1490, discuss the binding of organic ions by albumin. Klotz, in the Journal of the American Chemical Society, volume 68, 1946, pages 2299-2304, and Daniel and Weber, Biochem, 5:1893 (196) have demonstrated that spectral shifts and/or absorbance or flourescence changes accompany the interaction of albumin with an organic dye and that these changes could be partially reversed upon the addition of competing ligands.
Previous methods for the determination of fatty acids in aqueous samples include three principal techniques:
(1) Titration of the fatty acids with alkali, V. P. Dole, Journal of Clinical Investigation, 35:150 (1956).
(2) Determination as the copper salt after extraction with a suitable solvent, W. G. Duncombe, Biochemical Journal, 88:7 (1963), and
(3) Chromatographic procedures.
These techniques are time consuming and, in general, not adaptable to rapid, automated analysis techniques.
In addition to determining the free fatty acid content in aqueous samples, such as blood serum and milk, it has become desirable to determine the quantity of triglycerides, cholesterol esters and phospholipids present. It is of particular interest to analyze blood serum for both triglyceride and cholesterol content since excessive levels of these substances in the blood serum have been linked with a variety of cardiovascular problems. K. A. Slickers in Lab World, volume 27, pages 8-14 (1976), summarizes the enzymatic methods of determining the triglyceride content. In general, these techniques involve either the saponification of the triglyceride to glycerol with alkali hydroxide or its hydrolysis by a triacylglycerol hydrolase enzyme to free glycerol. The glycerol content then being determined by an appropriate technique. Komatsu in U.S. Pat. No. 3,898,130, describes an enzymatic hydrolysis process for triglycerides. Wahlfeld et al in U.S. Pat. No. 3,862,009 and Bucolo et al in U.S. Pat. No. 3,703,591 disclose integrated triglyceride determination processes. In this process, the triglyceride is enzymatically hydrolyzed to glycerol and the glycerol content determined spectrophotometrically by measuring the change in adsorption of the solution. In order to accomplish this, it was necessary for both Wahlfeld et al and Bucolo et al to include in their system adenosine triphosphate (ATP), glycerokinase (GK), phosphenol pyruvate (PEP), lactate dehydrogenase (LDH) and reduced nicotinamide-adenine-dinucleotide (NADH). The absorbance of this solution changes as NADH is oxidized to NAD as the end result of several enzymatic reactions.
In order to determine the quantity of cholesterol in a sample it is necessary to first hydrolyze any cholesterol esters to cholesterol. There are two basic techniques for accomplishing this. One is alcoholic saponification with an alkali hydroxide, such as KOH, and the other is enzymatic hydrolysis. The amount of total cholesterol may then be determined by a variety of techniques. E. V. Cardinal suggested in U.S. Pat. No. 3,479,154, precipitation of the cholesterol with known quantity of digitonin, separation of the precipitate, followed by analysis for digitonin.
Recently several integrated enzymatic processes for cholesterol determination have been proposed. Goodhue et al in U.S. Pat. No. 3,884,764, suggest enzymatically hydrolyzing the cholesterol ester with a combination of a lipase having cholesterol esterase activity, a protease and cholesterol oxidase whereby the cholesterol is converted to free cholesterol which is enzymatically oxidized. The quantity is then determined by measuring the quantity of one of the products of the cholesterol oxidation. The measurement may be conducted by enzymatic techniques if desired.
Methods for the determination of phospholipids, such as lecithin, sphingomyelin, cerebrosides, and gangliosides have proven unsatisfactory. Lecithin has been determined by procedures based upon selective extraction into appropriate organic solvents followed by chemical determination of organic soluble phosphorus. Chromatographic procedures have also been employed to isolate the lecithin fraction from interfering substances followed by chemical detection methods. The use of the enzymes phospholipase A.sub.2 and phospholipase D in the analysis for lecithin has been reported. Analytical methods for the determination of sphingomyelin, cerebrosides and gangliosides involve extraction, chromatographic separations to remove interfering substances, and the application of chemical detection methods. These techniques involve a number of processing steps which make such determinations time consuming and costly.
Accordingly, there exists a need for a technique which is capable of quantitatively and qualitatively analyzing aqueous media for free fatty acids and compounds which may be hydrolyzed to free fatty acids such as triglycerides, cholesterol esters and phospholipids in a simple manner. In particular, for a technique which allows the determination of the components by direct spectrophotometric or fluorometric means.